An AmotL2–Yap1 Module Integrates Flow and Junctional Mechanics to Specify Vascular Pruning Hotspots

Vascular networks constantly remodel to optimise blood flow, yet how endothelial cells (ECs) integrate haemodynamic cues with junctional mechanics to execute precise branch pruning remains unclear. Using live imaging and quantitative topology analysis in the zebrafish sub-intestinal vein plexus, we define the cellular events underlying flow-guided regression. Pruning occurs in geometrically disadvantaged, low-flow branches and is executed through transient polarity fluctuations, junctional remodelling and directed EC migration. We identify AmotL2 as an essential regulator of endothelial mechanical competence. AmotL2 maintains junctional architecture, actomyosin tension and appropriate cell packing, enabling ECs to polarise and rearrange in response to local flow differences. Loss of AmotL2 disrupts tension homeostasis, increases tissue compaction and prevents branch regression despite normal perfusion. In contrast, Yap1 plays an opposing, stabilising role, and its loss results in excessive pruning and network simplification. Together, AmotL2 and Yap1 constitute a mechanosensitive balancing module that interprets flow magnitude and vascular geometry to determine branch stability. This framework describes how mechanical forces sculpt vascular architecture during development and homeostasis.